Culture method for stable proliferation of pluripotent stem cell while maintaining undifferentiated state

ABSTRACT

Cultivating a pluripotent stem cell in a medium comprising at least one member selected from the group consisting of ethanolamine, an ethanolamine analog, and a pharmaceutically acceptable salt thereof, and which is substantially free of β-mercaptoethanol or contains β-mercaptoethanol at a concentration of not more than 9 μM, and the like, is effective for the proliferation of a pluripotent stem cell while maintaining an undifferentiated state.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/JP2013/085263, filed on Dec. 27, 2013, and claims priority toJapanese Patent Application No. 2013-016592, filed on Jan. 31, 2013,both of which are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to culture methods for the proliferationof pluripotent stem cells while maintaining an undifferentiated state,and particularly relates to a culture method for stable proliferation ofhuman pluripotent stem cells under serum-free, feeder-free conditionsand under single seeding conditions while maintaining anundifferentiated state and the like.

2. Discussion of the Background

Pluripotent stem cells such as ES (Embryonic stem) cells, iPS (inducedpluripotent stem) cells and the like are expected to be usable forregenerative medicine and the like in view of the superiorproliferativity and pluripotency. In particular, iPS cells areconsidered a highly superior material for regenerative medicine, sinceproduction and obtainment are relatively easy, there are few ethicalrestrictions for production, and further, from the aspect of rejectionin transplantation.

These pluripotent stem cells have conventionally been cultured byco-culture with a carrying cell (hereinafter a feeder cell) such as afibroblast and the like in a medium containing serum. For example, inCell, 2006, 126, 663-76, which is incorporated herein by reference inits entirety and which is the world-first report by Yamanaka et al. onthe production of iPS cells, iPS cells were established and maintainedand proliferated under conditions using a feeder cell and serum. Thesepluripotent stem cells proliferate while forming a colony whereindividual cells are clustered. When a colony is dissociated into singlecells and used for seeding (hereinafter single cell-seeding), the cellsbecome unstable. It is therefore a general practice to seed a colonymaintained to have a certain size (hereinafter colony seeding). Forexample, Nature Communications, 2012, 3:1236, which is incorporatedherein by reference in its entirety, discloses an example showing thatthe proliferation conditions of cells are prone to influence fromculture environment in the case of single cell-seeding compared tocolony seeding. In other words, single cell-seeding poses higher culturedifficulty than colony seeding.

To perform feeder-free culture, it is necessary to coat the bottom of aculture vessel with a substrate or scaffolding material replacing afeeder cell. As the substrate, an extracellular matrix component isoften used. JP-A-2011-78370, which is incorporated herein by referencein its entirety, discloses that use of an active fragment of laminin 511as a substrate is preferable for the proliferation of human ES/iPS cell,and single cell-seeding is also possible.

WO 2012/019122 and Nature Methods, 2011, 8, 424-429, both of which areincorporated herein by reference in their entireties, disclose thecomposition of a serum-free medium for human pluripotent stem cells.This composition called E8 contains DMEM/F12 as a basal medium, andfurther contains some factors such as bFGF, insulin and the like. Atpresent, it is considered the minimum composition for cultivating humanpluripotent stem cells.

Ethanolamine is known to contribute to the promoted proliferation ofmesenchymal stem cells when used as an additive in a medium. Forexample, patent document 3 patent document 3: JP-A-2006-325445, which isincorporated herein by reference in its entirety, discloses an examplesuggesting that ethanolamine promotes proliferation of mesenchymal stemcells.

In addition, JP-A-2009-542247, which is incorporated herein by referencein its entirety, describes a method of maintaining primate embryonicstem cells in a medium containing ethanolamine, 2-mercaptoethanol, acomplex of oleic acid with fatty acid-free bovine albumin, heparin andthe like, and the like, and WO 2005/063968, which is incorporated hereinby reference in its entirety, describes a medium for culturing ES cells,which contains 2-mercaptoethanol, 2-ethanolamine, a complex of oleicacid with fatty acid-free bovine serum albumin and the like. U.S. Pat.No. 8,569,061, which is incorporated herein by reference in itsentirety, discloses a medium for ES cells, which contains human albumin,ethanolamine, β-mercaptoethanol and the like.

The above-mentioned JP-A-2009-542247, WO 2005/063968, and U.S. Pat. No.8,569,061 describe given amounts (10 μM, 10 μM, 100 μM, respectively) of2-mercaptoethanol (β-mercaptoethanol) as an essential component ofmedium. In JP-A-2009-542247, oleic acid is added to bovine albumin afterremoval of fatty acid, such that the bovine albumin carries oleic acid,and 9.4 mg/g of oleic acid is added to albumin.

In the meantime, it has been reported heretofore that sulfatedpolysaccharides have an effect to protect growth factors fromdegradation, denaturation, inactivation and the like. For example, WO92/13526, which is incorporated herein by reference in its entirety,discloses that carrageenan stabilizes bFGF, and describes in theExamples that a protecting agent containing sulfated polysaccharidessuch as heparin, dextran sulfate, carrageenan and the like protects bFGFfrom hydrolysis and heat denaturation. However, an effect provided by acombination of ethanolamine and sulfated polysaccharides is notdisclosed. Moreover, the above-mentioned JP-A-2009-542247 discloses amedium integrally containing ethanolamine and heparin; however, adetailed effect of each of them has not been known to date.

SUMMARY OF THE INVENTION

The above-mentioned conventional culture methods pose various problemswhen practicing a regenerative medicine using a pluripotent stem cells,particularly at an industrial level. As a feeder cell, xenogenic cellssuch as mouse embryo-derived fibroblast and the like are generally used,and the problem of safety after transplantation has been pointed out.Also, from the aspects of cost and cell quality management, culturewithout using a feeder cell (hereinafter feeder-free culture) ispreferable. As for serum, the problem of infection source, and concernabout property difference between lots which causes inconsistent cultureresults have been pointed out. As for the colony seeding, the number ofseeded cells cannot be adjusted rigorously, which makes it difficult tomanage culture schedule, and produces personal variation in the cultureresults. To produce pluripotent stem cells for a regenerative medicineat an industrial level, the work needs to be performed by plural workersunder the conditions where procedures and schedule are rigorouslymanaged. Moreover, since β-mercaptoethanol, which is generally added toa medium for pluripotent stem cell, is designated as toxin, it ispreferable to not add the same to a medium or reduce the amount thereofto be added as much as possible, considering the complicated handlingand the like. The influence of its amount to be added on the culture ofpluripotent stem cells has not been clarified in detail yet.

From the foregoing, there is a demand for the development of a culturemethod, which adopts serum-free, feeder-free and single cell-seeding ofpluripotent stem cells, and is different from conventional culturemethods. In addition, cost is a major issue at an industrial level.Therefore, a mere success in the performance of serum-free, feeder-freeand single cell-seeding culture is not sufficient, and such cultureaffording the largest possible number of cells per unit time, namely,culture with superior proliferation efficiency, is desirable.

The present inventors tried serum-free, feeder-free and singlecell-seeding culture by a conventional culture method using the activefragment of laminin 511 described in JP-A-m 2011-78370 as a substrateand a medium having the E8 composition. However, they could not performstable maintenance culture for a long term. It is necessary to elucidateand overcome the cause of such instability in culture, and furtherimprove the proliferative ability.

It is therefore an object of the present invention to provide a meansfor proliferating pluripotent stem cells while maintaining anundifferentiated state. A further object of the present invention is toprovide a means for stably and highly efficiently proliferatingpluripotent stem cells while maintaining an undifferentiated state, inserum-free, feeder-free and single cell-seeding culture.

Means of Solving the Problems

The present inventors have conducted intensive studies in an attempt tosolve the aforementioned problems and found that a feeder cell releasesethanolamine, and ethanolamine promotes the proliferation of pluripotentstem cells. They have also found that E8 causes remarkable propertydegradation after thawing and preparation, which may possibly causeinstability of culture using E8, and further found that albumin cansuppress such property degradation. They have also found that albuminnot only has a medium stabilizing effect but also has an action topotentiate the above-mentioned cell proliferation effect ofethanolamine. They have also found that sulfated saccharides have aproliferation promoting and medium stabilizing effect in the presence ofethanolamine. The present inventors further showed that the amounts of(3-mercaptoethanol, and fatty acid carried by albumin influenceproliferation of pluripotent stem cells while maintaining anundifferentiated state. The present inventors have conducted furtherstudies based on these findings and completed the present invention.

Accordingly, the present invention is as described below.

(1) A culture method for a proliferation of a pluripotent stem cellwhile maintaining an undifferentiated state, comprising a step ofcultivating the pluripotent stem cell in a medium added with at leastone selected from the group consisting of ethanolamine, ethanolamineanalogs and pharmaceutically acceptable salts thereof, and substantiallyfree of β-mercaptoethanol or containing β-mercaptoethanol at aconcentration of not more than 9 μM.

(2) The method of (1), wherein the concentration of at least oneselected from the group consisting of ethanolamine, ethanolamine analogsand pharmaceutically acceptable salts thereof in the medium is 1 μM-1000μM.

(3) The method of (1), wherein the concentration of at least oneselected from the group consisting of ethanolamine, ethanolamine analogsand pharmaceutically acceptable salts thereof in the medium is 5 μM-200μM.

(4) The method of any of (1)-(3), wherein the ethanolamine analog is acompound represented by the following formula

X—CH₂—CH₂—O—Y

whereinX is R¹—N(R²)— [R¹ and R² are the same or different and each is ahydrogen atom or an amino-protecting group] or R³—CH—N— [R³—CH is H—CHor a Shiff base type amino-protecting group]; andY is —P(═O)(OH)—O—R⁴ [R⁴ is —CH₂—CH(O—R⁵)—CH₂—O—R⁶ (R⁵ and R⁶ are thesame or different and each is an acyl group having 2-30 carbon atoms ora hydrogen atom) or a hydrogen atom], a hydrogen atom or ahydroxy-protecting group.

(5) The method of (4), wherein R¹ and R² are the same or different andeach is a hydrogen atom, a halogen atom, a hydroxy group, an aryl group,an acyl group having 2-30 carbon atoms, an alkyl group having 1-6 carbonatoms, an alkoxyl group having 1-6 carbon atoms, a hydroxyalkyl grouphaving 1-6 carbon atoms, a haloalkyl group having 1-6 carbon atoms, ahaloalkoxyl group having 1-6 carbon atoms or a halohydroxyalkyl grouphaving 1-6 carbon atoms, and R³ is a hydrogen atom, a halogen atom, ahydroxy group, an aryl group, an acyl group having 2-30 carbon atoms, analkyl group having 1-6 carbon atoms, an alkoxyl group having 1-6 carbonatoms, a hydroxyalkyl group having 1-6 carbon atoms, a haloalkyl grouphaving 1-6 carbon atoms, a haloalkoxyl group having 1-6 carbon atoms ora halohydroxyalkyl group having 1-6 carbon atoms.

(6) The method of any of (1)-(3), wherein the ethanolamine analog is oneor plural selected from the group consisting of phosphoethanolamine,monomethylethanolamine, dimethylethanolamine,N-acylphosphatidylethanolamine, phosphatidylethanolamine, andlysophosphatidylethanolamine.

(7) The method of any of (1)-(6), wherein the medium is further addedwith albumin.

(8) The method of (7), wherein the concentration of the albumin in themedium is 0.1 g/l-20 g/l.

(9) The method of (7), wherein the concentration of the albumin in themedium is 1 g/l-8 g/l.

(10) The method of any of (7)-(9), wherein the albumin is obtained fromplasma of an animal (including human) or by gene recombinationtechnology.

(11) The method of any of (7)-(10), wherein the amount of fatty acidcarried by the albumin in the medium is not more than 9 mg/g.

(12) The method of any of (7)-(10), wherein the amount of fatty acidcarried by the albumin in the medium is not more than 2.2 mg/g.

(13) The method of any of (1)-(12), wherein the medium is further addedwith sulfated saccharide and/or a pharmaceutically acceptable saltthereof.

(14) The method of (13), wherein the concentration of the sulfatedsaccharide and/or a pharmaceutically acceptable salt thereof in themedium is 1-1000 ng/ml.

(15) The method of (13) or (14), wherein the sulfated saccharide or apharmaceutically acceptable salt thereof is dextran sulfate Na having anaverage molecular weight of 2,500-7,500.

(16) A culture method for a proliferation of a pluripotent stem cellwhile maintaining an undifferentiated state, comprising a step ofcultivating the pluripotent stem cell in a medium added with at leastone selected from the group consisting of ethanolamine, ethanolamineanalogs and pharmaceutically acceptable salts thereof, and added withsulfated saccharide and/or a pharmaceutically acceptable salt thereof.

(17) The method of (16), wherein the concentration of the sulfatedsaccharide and/or a pharmaceutically acceptable salt thereof in themedium is 1-1000 ng/ml.

(18) The method of (16) or (17), wherein the sulfated saccharide or apharmaceutically acceptable salt thereof is dextran sulfate Na having anaverage molecular weight of 2,500-7,500.

(19) The method of any of (16)]-(18), wherein the medium is furtheradded with albumin.

(20) The method of (19), wherein the concentration of the albumin in themedium is 0.1 g/l-20 g/l.

(21) The method of (19), wherein the concentration of the albumin in themedium is 1 g/l-8 g/l.

(22) The method of any of (19)-(21), wherein the albumin is obtainedfrom plasma of an animal (including human) or by gene recombinationtechnology.

(23) The method of any of (1)-(22), wherein the culture is performed inthe absence of a feeder cell.

(24) The method of (23), wherein the culture is performed by using anextracellular matrix or an active fragment thereof, or an artificialproduct mimicking the function thereof.

(25) The method of (23), wherein the culture is performed by usinglaminin 511 or an active fragment thereof or matrigel.

(26) The method of any of (1)-(25), wherein the culture is performed bysingle cell-seeding.

(27) The method of any of (1)-(26), wherein the culture is performedunder serum-free conditions.

(28) The method of any of (1)-(27), wherein the medium is substantiallyfree of a component derived from an animal other than human.

(29) The method of any of (1)-(28), wherein the pluripotent stem cell isan embryonic stem cell (ES cell) or induced pluripotent stem cell (iPScell).

(30) The method of any of (1)-(29), wherein the pluripotent stem cell isderived from primates.

(31) The method of any of (1)-(30), wherein the pluripotent stem cell ishuman iPS cell.

(32) A preservation stabilizing method for a medium for a proliferationof a pluripotent stem cell, comprising adding at least one selected fromthe group consisting of ethanolamine, ethanolamine analogs andpharmaceutically acceptable salts thereof, and adding sulfatedsaccharide and/or a pharmaceutically acceptable salt thereof.

(33) The method of (32), wherein the final concentration of the sulfatedsaccharide and/or a pharmaceutically acceptable salt thereof in use is1-1000 ng/ml.

(34) The method of (32) or (33), further comprising adding albumin.

(35) The method of (34), wherein the final concentration of the albuminin use is 0.1 g/l-20 g/l.

(36) The method of [34], wherein the final concentration of the albuminin use is 1 g/l-8 g/l.

(37) The method of any of (34)-(36), wherein the albumin is obtainedfrom plasma of an animal (including human) or by gene recombinationtechnology.

(38) The method of any of (34)-(37), wherein the amount of fatty acidcarried by the albumin is not more than 9 mg/g.

(39) The method of any of (34)-(37), wherein the amount of fatty acidcarried by the albumin is not more than 2.2 mg/g.

(40) The method of any of (32)-(39), wherein the medium is substantiallyfree of β-mercaptoethanol or contains (3-mercaptoethanol at a finalconcentration of not more than 9 μM.

(41) The method of any of (32)-(40), wherein the medium is for aproliferation of a pluripotent stem cell while maintaining anundifferentiated state.

(42) A medium additive for a proliferation of a pluripotent stem cellwhile maintaining an undifferentiated state, comprising at least oneselected from the group consisting of ethanolamine, ethanolamine analogsand pharmaceutically acceptable salts thereof, and substantially free ofβ-mercaptoethanol or containing β-mercaptoethanol at a concentration ofnot more than 9 μM when in use.

(43) The medium additive of (42), wherein the ethanolamine analog is acompound represented by the following formula

X—CH₂—CH₂—O—Y

whereinX is R¹—N(R²)— [R¹ and R² are the same or different and each is ahydrogen atom or an amino-protecting group] or R³—CH═N— [R³—CH is H—CHor a Shiff base type amino-protecting group]; andY is —P(═O) (OH)—O—R⁴ [R⁴ is —CH₂—CH(O—R⁵)—CH₂—O—R⁶ (R⁵ and R⁶ are thesame or different and each is an acyl group having 2-30 carbon atoms ora hydrogen atom) or a hydrogen atom], a hydrogen atom or ahydroxy-protecting group.

(44) The medium additive of (43), wherein R¹ and R² are the same ordifferent and each is a hydrogen atom, a halogen atom, a hydroxy group,an aryl group, an acyl group having 2-30 carbon atoms, an alkyl grouphaving 1-6 carbon atoms, an alkoxyl group having 1-6 carbon atoms, ahydroxyalkyl group having 1-6 carbon atoms, a haloalkyl group having 1-6carbon atoms, a haloalkoxyl group having 1-6 carbon atoms or ahalohydroxyalkyl group having 1-6 carbon atoms, and R³ is a hydrogenatom, a halogen atom, a hydroxy group, an aryl group, an acyl grouphaving 2-30 carbon atoms, an alkyl group having 1-6 carbon atoms, analkoxyl group having 1-6 carbon atoms, a hydroxyalkyl group having 1-6carbon atoms, a haloalkyl group having 1-6 carbon atoms, a haloalkoxylgroup having 1-6 carbon atoms or a halohydroxyalkyl group having 1-6carbon atoms.

(45) The medium additive of any of (42)-(44), wherein the ethanolamineanalog is one or plural selected from the group consisting ofphosphoethanolamine, monomethylethanolamine, dimethylethanolamine,N-acylphosphatidylethanolamine, phosphatidylethanolamine, andlysophosphatidylethanolamine.

(46) The medium additive of any of (42)-(45), further comprisingalbumin.

(47) The medium additive of (46), wherein the final concentration of thealbumin in use is 0.1 g/l-20 g/l.

(48) The medium additive of (46), wherein the final concentration of thealbumin in use is 1 g/l-8 g/l.

(49) The medium additive of any of (46)-(48), wherein the amount offatty acid carried by the albumin is not more than 9 mg/g.

(50) The medium additive of any of (46)-(48), wherein the amount offatty acid carried by the albumin is not more than 2.2 mg/g.

(51) The medium additive of any of (46)-(50), wherein the albumin isobtained from plasma of an animal (including human) or by generecombination technology.

(52) The medium additive of any of (42)-(51), further comprisingsulfated saccharide and/or a pharmaceutically acceptable salt thereof.

(53) The medium additive of (52), wherein the final concentration of thesulfated saccharide and/or a pharmaceutically acceptable salt thereof inuse is 1-1000 ng/ml.

(54) The medium additive of (52) or (53), wherein the sulfatedsaccharide is at least one selected from the group consisting ofsulfated monosaccharide, sulfated disaccharide, sulfated polysaccharide,sulfated sugar alcohol and sulfated cyclitol.

(55) The medium additive of any of (52)-(54), wherein the aforementionedsulfated saccharide or a pharmaceutically acceptable salt thereof is atleast one selected from the group consisting of dextran sulfate Na,cellulose SO₃Na, xanthan gum SO₃Na, fucoidan, alginate SO₃Na, inulinSO₃Na, maltoheptaose SO₃Na, stachyose SO₃Na, maltotriose SO₃Na, multitolSO₃Na, sucrose8SO₃K, glucose SO₃Na, myo-6 inositol SO₃K, α-cyclodextrinSO₃Na, mannitol SO₃Na, xylitol SO₃Na and erythritol SO₃Na.

(56) The medium additive of [55], wherein the aforementioned sulfatedsaccharide or a pharmaceutically acceptable salt thereof is at least oneselected from the group consisting of dextran sulfate Na, fucoidan,maltoheptaose SO₃Na, maltotriose SO₃Na, multitol SO₃Na and sucrose8SO₃K.

(57) The medium additive of any of (52)-(56), wherein the aforementionedsulfated saccharide or a pharmaceutically acceptable salt thereof isdextran sulfate Na having an average molecular weight of 2,500-7,500.

(58) The medium additive of any of (42)-(57), wherein the proliferationof a pluripotent stem cell while maintaining an undifferentiated stateis performed under the conditions without using a feeder cell.

(59) The medium additive of (58), wherein the conditions without using afeeder cell do not include use of a feeder cell but include use of anextracellular matrix or an active fragment thereof, or an artificialproduct mimicking the function thereof.

(60) The medium additive of (58), wherein the conditions without using afeeder cell do not include use of a feeder cell but include use oflaminin 511 or an active fragment thereof or matrigel.

(61) The medium additive of any of (42)-(60), wherein the proliferationof a pluripotent stem cell while maintaining an undifferentiated stateis performed by single cell-seeding.

(62) The medium additive of any of (42)-(61), wherein the proliferationof a pluripotent stem cell while maintaining an undifferentiated stateis performed under serum-free conditions.

(63) The medium additive of any of (42)-(62), comprising substantiallyfree of a component derived from an animal other than human.

(64) The medium additive of any of (42)-(63), wherein the pluripotentstem cell is an embryonic stem cell (ES cell) or induced pluripotentstem cell (iPS cell).

(65) The medium additive of any of (42)-(64), wherein the pluripotentstem cell is derived from primates.

(66) The medium additive of any of (42)-(65), wherein the pluripotentstem cell is human iPS cell.

(67) A medium for proliferation of a pluripotent stem cell whilemaintaining an undifferentiated state, which comprises the mediumadditive of any of (42)-(66).

(68) A culture method for a proliferation of an induced pluripotent stemcell (iPS cell) while maintaining an undifferentiated state, comprisinga step of cultivating the induced pluripotent stem cell (iPS cell) in amedium added with at least one selected from the group consisting ofethanolamine, ethanolamine analogs and pharmaceutically acceptable saltsthereof.

(69) The method of (68), wherein the concentration of at least oneselected from the group consisting of ethanolamine, ethanolamine analogsand pharmaceutically acceptable salts thereof in the medium is 1 μM-1000μM.

(70) The method of (68), wherein the concentration of at least oneselected from the group consisting of ethanolamine, ethanolamine analogsand pharmaceutically acceptable salts thereof in the medium is 5 μM-200μM.

(71) The method of any of (68)-(70), wherein the ethanolamine analog isa compound represented by the following formula

X—CH₂—CH₂—O—Y

whereinX is R¹—N(R²)— [R¹ and R² are the same or different and each is ahydrogen atom or an amino-protecting group] or R³—CH═N— [R³—CH is H—CHor a Shiff base type amino-protecting group]; andY is —P(═O) (OH)—O—R⁴ [R⁴ is —CH₂—CH(O—R⁵)—CH₂—O—R⁶ (R⁵ and R⁶ are thesame or different and each is an acyl group having 2-30 carbon atoms ora hydrogen atom) or a hydrogen atom], a hydrogen atom or ahydroxy-protecting group.

(72) The method of (71), wherein R¹ and R² are the same or different andeach is a hydrogen atom, a halogen atom, a hydroxy group, an aryl group,an acyl group having 2-30 carbon atoms, an alkyl group having 1-6 carbonatoms, an alkoxyl group having 1-6 carbon atoms, a hydroxyalkyl grouphaving 1-6 carbon atoms, a haloalkyl group having 1-6 carbon atoms, ahaloalkoxyl group having 1-6 carbon atoms or a halohydroxyalkyl grouphaving 1-6 carbon atoms, and R³ is a hydrogen atom, a halogen atom, ahydroxy group, an aryl group, an acyl group having 2-30 carbon atoms, analkyl group having 1-6 carbon atoms, an alkoxyl group having 1-6 carbonatoms, a hydroxyalkyl group having 1-6 carbon atoms, a haloalkyl grouphaving 1-6 carbon atoms, a haloalkoxyl group having 1-6 carbon atoms ora halohydroxyalkyl group having 1-6 carbon atoms.

(73) The method of any of (68)-(72), wherein the ethanolamine analog isone or plural selected from the group consisting of phosphoethanolamine,monomethylethanolamine, dimethylethanolamine,N-acylphosphatidylethanolamine, phosphatidylethanolamine, andlysophosphatidylethanolamine.

(74) The method of any of (68)-(73), wherein the medium is further addedwith albumin.

(75) The method of (74), wherein the concentration of the albumin in themedium is 0.1 g/l-20 g/l.

(76) The method of (74), wherein the concentration of the albumin in themedium is 1 g/l-8 g/l.

(77) The method of any of (74)-(76), wherein the albumin is obtainedfrom plasma of an animal (including human) or by gene recombinationtechnology.

(78) The method of any of (74)-(77), wherein the amount of fatty acidcarried by the albumin in the medium is not more than 9 mg/g.

(79) The method of any of (74)-(77), wherein the amount of fatty acidcarried by the albumin in the medium is not more than 2.2 mg/g.

(80) The method of any of (68)-(79), wherein the culture is performed inthe absence of a feeder cell.

(81) The method of (80), wherein the culture is performed by using anextracellular matrix or an active fragment thereof, or an artificialproduct mimicking the function thereof.

(82) The method of (80), wherein the culture is performed by usinglaminin 511 or an active fragment thereof or matrigel.

(83) The method of any of (68)-(82), wherein the culture is performed bysingle cell-seeding.

(84) The method of any of (68)-(83), wherein the culture is performedunder serum-free conditions.

(85) The method of any of (68)-(84), wherein the medium is substantiallyfree of a component derived from an animal other than human.

(86) The method of any of (68)-(85), wherein the induced pluripotentstem cell is derived from primates.

(87) The method of any of (68)-(86), wherein the induced pluripotentstem cell is human iPS cell.

(88) A preservation stabilizing method for a medium for a proliferationof a pluripotent stem cell, comprising adding at least one selected fromthe group consisting of ethanolamine, ethanolamine analogs andpharmaceutically acceptable salts thereof.

(89) The method of (88), further comprising adding sulfated saccharideand/or a pharmaceutically acceptable salt thereof.

(90) The method of (89), wherein the final concentration of the sulfatedsaccharide and/or a pharmaceutically acceptable salt thereof in use is1-1000 ng/ml.

(91) The method of any of (88)-(90), further comprising adding albumin.

(92) The method of (91), wherein the final concentration of the albuminin use is 0.1 g/l-20 g/l.

(93) The method of (91), wherein the final concentration of the albuminin use is 1 g/l-8 g/l.

(94) The method of any of (91)-(93), wherein the albumin is obtainedfrom plasma of an animal (including human) or by gene recombinationtechnology.

(95) The method of any of (91)-(94), wherein the amount of fatty acidcarried by the albumin is not more than 9 mg/g.

(96) The method of any of (91)-(94), wherein the amount of fatty acidcarried by the albumin is not more than 2.2 mg/g.

(97) The method of any of (88)-(96), wherein the medium is substantiallyfree of β-mercaptoethanol or contains β-mercaptoethanol at a finalconcentration of not more than 9 μM.

(98) The method of any of (88)-(97), wherein the medium is for aproliferation of a pluripotent stem cell while maintaining anundifferentiated state.

(99) A medium additive for a proliferation of a pluripotent stem cellwhile maintaining an undifferentiated state, comprising at least oneselected from the group consisting of ethanolamine, ethanolamine analogsand pharmaceutically acceptable salts thereof.

(100) The medium additive of [99], wherein the ethanolamine analog is acompound represented by the following formula

X—CH₂—CH₂—O—Y

whereinX is R¹—N(R²)— [R¹ and R² are the same or different and each is ahydrogen atom or an amino-protecting group] or R³—CH═N— [R³—CH is H—CHor a Shiff base type amino-protecting group]; andY is —P(═O) (OH)—O—R⁴ [R⁴ is —CH₂—CH(O—R⁵)—CH₂—O—R⁶ (R⁵ and R⁶ are thesame or different and each is an acyl group having 2-30 carbon atoms ora hydrogen atom) or a hydrogen atom], a hydrogen atom or ahydroxy-protecting group.

(101) The medium additive of (100), wherein R¹ and R² are the same ordifferent and each is a hydrogen atom, a halogen atom, a hydroxy group,an aryl group, an acyl group having 2-30 carbon atoms, an alkyl grouphaving 1-6 carbon atoms, an alkoxyl group having 1-6 carbon atoms, ahydroxyalkyl group having 1-6 carbon atoms, a haloalkyl group having 1-6carbon atoms, a haloalkoxyl group having 1-6 carbon atoms or ahalohydroxyalkyl group having 1-6 carbon atoms, and R³ is a hydrogenatom, a halogen atom, a hydroxy group, an aryl group, an acyl grouphaving 2-30 carbon atoms, an alkyl group having 1-6 carbon atoms, analkoxyl group having 1-6 carbon atoms, a hydroxyalkyl group having 1-6carbon atoms, a haloalkyl group having 1-6 carbon atoms, a haloalkoxylgroup having 1-6 carbon atoms or a halohydroxyalkyl group having 1-6carbon atoms.

(102) The medium additive of any of (99)-(101), wherein the ethanolamineanalog is one or plural selected from the group consisting ofphosphoethanolamine, monomethylethanolamine, dimethylethanolamine,N-acyiphosphatidylethanolamine, phosphatidylethanolamine, andlysophosphatidylethanolamine.

(103) The medium additive of any of (99)-(102), further comprisingalbumin.

(104) The medium additive of (103), wherein the albumin is obtained fromplasma of an animal (including human) or by gene recombinationtechnology.

(105) The medium additive of any of (99)-(104), further comprisingsulfated saccharide and/or a pharmaceutically acceptable salt thereof.

(106) The medium additive of (105), wherein the final concentration ofthe sulfated saccharide and/or a pharmaceutically acceptable saltthereof in use is 1-1000 ng/ml.

(107) The medium additive of (105) or (106), wherein the sulfatedsaccharide is at least one selected from the group consisting ofsulfated monosaccharide, sulfated disaccharide, sulfated polysaccharide,sulfated sugar alcohol and sulfated cyclitol.

(108) The medium additive of any of (105)-(107), wherein theaforementioned sulfated saccharide or a pharmaceutically acceptable saltthereof is at least one selected from the group consisting of dextransulfate Na, cellulose SO₃Na, xanthan gum SO₃Na, fucoidan, alginateSO₃Na, inulin SO₃Na, Maltoheptaose SO₃Na, stachyose SO₃Na, maltotrioseSO₃Na, multitol SO₃Na, sucrose8SO₃K, glucose SO₃Na, myo-6 inositol SO₃K,α-cyclodextrin SO₃Na, mannitol SO₃Na, xylitol SO₃Na and erythritolSO₃Na.

(109) The medium additive of (108), wherein the aforementioned sulfatedsaccharide or a pharmaceutically acceptable salt thereof is at least oneselected from the group consisting of dextran sulfate Na, fucoidan,Maltoheptaose SO₃Na, maltotriose SO₃Na, multitol SO₃Na and sucrose8SO₃K.

(110) The medium additive of any of (105)-(109), wherein theaforementioned sulfated saccharide or a pharmaceutically acceptable saltthereof is dextran sulfate Na having an average molecular weight of2,500-7,500.

(111) The medium additive of any of (99)-(110), wherein theproliferation of a pluripotent stem cell while maintaining anundifferentiated state is performed under the conditions without using afeeder cell.

(112) The medium additive of (111), wherein the conditions without usinga feeder cell do not include use of a feeder cell but include use of anextracellular matrix or an active fragment thereof, or an artificialproduct mimicking the function thereof.

(113) The medium additive of (111), wherein the conditions without usinga feeder cell do not include use of a feeder cell but include use oflaminin 511 or an active fragment thereof or matrigel.

(114) The medium additive of any of (99)-(113), wherein theproliferation of a pluripotent stem cell while maintaining anundifferentiated state is performed by single cell-seeding.

(115) The medium additive of any of (99)-(114), wherein theproliferation of a pluripotent stem cell while maintaining anundifferentiated state is performed under serum-free conditions.

(116) The medium additive of any of (99)-(115), comprising substantiallyfree of a component derived from an animal other than human.

(117) The medium additive of any of (99)-(116), wherein the pluripotentstem cell is an embryonic stem cell (ES cell) or induced pluripotentstem cell (iPS cell).

(118) The medium additive of any of (99)-(117), wherein the pluripotentstem cell is derived from primates.

(119) The medium additive of any of (99)-(118), wherein the pluripotentstem cell is human iPS cell.

(120) A medium for proliferation of a pluripotent stem cell whilemaintaining an undifferentiated state, which comprises the mediumadditive of any of (99)-(119).

Effect of the Invention

According to the present invention, pluripotent stem cells can be stablyand efficiently proliferated, and can be stably proliferated for a longterm while maintaining an undifferentiated state even in serum-free,feeder-free and single cell-seeding culture.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same become betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows a medium stabilizing effect by albumin under preservationconditions at 4° C.

FIG. 2 shows a cell proliferation promoting effect of ethanolamine andan effect of combination with albumin.

FIG. 3 shows a cell proliferation promoting effect by a combination ofethanolamine and dextran sulfate sodium, which is expressed by acumulative viable cell increase rate (fold).

FIG. 4 shows the results of alkaline phosphatase staining of iPS cellcolonies after long-term culture in an E8 minimum composition mediumadded with albumin, ethanolamine and dextran sulfate sodium.

FIG. 5 shows a cell proliferation promoting effect of ethanolamine inculture using matrigel as a basal lamina matrix.

FIG. 6 shows that medium change can be omitted in culture of iPS cells,by adding dextran sulfate sodium to a medium containing albumin andethanolamine.

FIG. 7 shows a medium stabilizing effect of a combination ofethanolamine and dextran sulfate sodium under room temperaturepreservation conditions.

FIG. 8 shows a cell proliferation promoting effect of O-phosphorylethanolamine.

FIG. 9 shows a cell proliferation promoting effect of2-(methylamino)ethanol.

FIG. 10 shows a cell proliferation promoting effect of2-dimethylaminoethanol.

FIG. 11 shows a cell proliferation promoting effect of ethanolaminehydrochloride.

FIG. 12 shows a cell proliferation suppressive effect along with anincrease in the amount of oleic acid carried by albumin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Culture Method for Proliferation of Pluripotent Stem Cells whileMaintaining an Undifferentiated State.

The present invention provides a culture method for the proliferation ofa pluripotent stem cell while maintaining an undifferentiated state,comprising a step of cultivating a pluripotent stem cell in a mediumadded with at least one selected from the group consisting ofethanolamine, ethanolamine analogs and pharmaceutically acceptable saltsthereof, and substantially free of β-mercaptoethanol or containingβ-mercaptoethanol at a concentration of not more than 9 μM (hereinafterto be also referred to as the culture method of the present invention).

The present invention is based on the new finding that a feeder cellreleases ethanolamine, and addition of ethanolamine can promoteproliferation of pluripotent stem cells while maintaining anundifferentiated state even in feeder-free culture. As a feeder cell,xenogenic cells are generally used, and incidence of infection with afeeder cell-derived virus is also known. The present invention can solvethese problems, and is extremely useful in the field of regenerativemedicine.

Also, the present invention is based on the finding that pluripotentstem cells can be proliferated well while maintaining anundifferentiated state even when the concentration of β-mercaptoethanolin the medium is reduced. In the culture method of the presentinvention, the medium is preferably substantially or completely free ofβ-mercaptoethanol. Even when the medium contains β-mercaptoethanol, itsconcentration is preferably not more than 9 μM.

In the present specification, being “substantially free ofβ-mercaptoethanol” means that the content concentration ofβ-mercaptoethanol is below detection limit.

In the present invention, the “pluripotent stem cell” means an immaturecell having self-renewal capacity and differentiation/proliferationcapacity, which is capable of differentiating into any tissue or cellconstituting living organisms. Examples of the pluripotent stem cellinclude embryonic stem cells (ES cells), embryonic germ cells (EGcells), induced pluripotent stem cells (iPS cells) and the like. A stemcell established by cultivating an early embryo generated by nucleartransplantation of the nucleus of a somatic cell is also included in thepluripotent stem cell (Nature, 385, 810 (1997); Science, 280, 1256(1998); Nature Biotechnology, 17, 456 (1999); Nature, 394, 369 (1998);Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984(1999); Nature Genetics, 24, 109 (2000), all of which are incorporatedherein by reference in their entireties). The pluripotent stem cell inthe present invention does not include a multipotent stem cell. Themultipotent stem cell means a cell capable of differentiating intoplural, though not all, types of tissues and cells and includes somaticstem cells such as mesenchymal stem cell and the like.

While the culture method of the present invention can be preferably usedfor any pluripotent stem cells, it is preferably used for proliferationof embryonic stem cells (ES 55 cells) or induced pluripotent stem cells(iPS cells) while maintaining an undifferentiated state.

Also, the culture method of the present invention can be preferably usedfor pluripotent stem cells derived from any animals. The pluripotentstem cells cultured by using the medium of the present invention are,for example, pluripotent stem cells derived from rodents such as mouse,rat, hamster, guinea pig and the like, Lagomorpha such as rabbit and thelike, Ungulata such as swine, bovine, goat, horse, sheep and the like,Carnivora such as dog, cat and the like, primates such as human, monkey,Macaca mulatta, marmoset, orangutan, chimpanzee and the like. Preferredare pluripotent stem cells derived from primates. When it is used forregenerative medicine, human iPS cells are preferable.

In the present invention, the “proliferation while maintaining anundifferentiated state” of pluripotent stem cells means that pluripotentstem cells can proliferate in an undifferentiated state whilemaintaining the pluripotency. That is, the culture method of the presentinvention can also be said to be a method of proliferating pluripotentstem cells in an undifferentiated state while maintaining pluripotency.Whether a pluripotent stem cell is maintained in an undifferentiatedstate is confirmed by alkaline phosphatase staining as shown in thebelow-mentioned Examples. Stained cells are evaluated as beingmaintained in an undifferentiated state.

The “ethanolamine” (also called 2-aminoethanol, monoethanolamine) usedin the present invention may be isolated and purified from a naturalproduct or a processed product thereof, or a synthesized product.Ethanolamine can be produced by reacting ethylene oxide and ammonia.Ethanolamine can also be isolated and purified from a natural product ora processed product thereof by known techniques such as solventextraction, various chromatographys and the like. Ethanolamine may be acommercially available product and can be obtained from, for example,Sigma-Aldrich Co., Ltd. and the like.

Examples of the “ethanolamine analog” to be used in the presentinvention include a compound represented by the following formula

X—CH₂—CH₂—O—Y

whereinX is R¹—N(R²)— (R¹ and R² are the same or different and each is ahydrogen atom or an amino-protecting group) or R³—CH—N— (R³—CH is H—CHor a Shiff base type amino-protecting group); andY is —P(═O)(OH)—O—R⁴ (R⁴ is —CH₂—CH(O—R⁵)—CH₂—O—R⁶ (R⁵ and R⁶ are thesame or different and each is an acyl group having 2 to 30 carbon atomsor a hydrogen atom) or a hydrogen atom), a hydrogen atom or ahydroxy-protecting group].

As for the “amino-protecting group”, for example, books such as Green etal., Protective Groups in Organic Synthesis, 3rd Edition, 1999, JohnWiley & Sons, Inc., which is incorporated herein by reference in itsentirety, and the like can be referred to, and an appropriate protectinggroup can be selected, introduced and removed. Examples of the“amino-protecting group” include a halogen atom, a hydroxy group, anaryl group, an acyl group having 2 to 30 carbon atoms, an alkyl grouphaving 1 to 6 carbon atoms, an alkoxyl group having 1 to 6 carbon atoms,a hydroxyalkyl group having 1 to 6 carbon atoms, a haloalkyl grouphaving 1 to 6 carbon atoms, a haloalkoxyl′ group having 1 to 6 carbonatoms or a halohydroxyalkyl group having 1 to 6 carbon atoms.Furthermore, a leaving group that can be bound to an amino group forforming a prodrug can also be mentioned.

The “halogen atom” means a fluorine atom, a chlorine atom, a bromineatom, or an iodine atom.

Examples of the “aryl group” include phenyl, 1-naphthyl, 2-naphthyl andthe like.

Examples of the “acyl group having 2 to 30 carbon atoms” include asaturated carboxylic acid acyl group and an unsaturated carboxylic acidacyl group. Examples of the saturated carboxylic acid acyl group includeacetyl(ethanoyl), propanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl,octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl,tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl,nonadecanoyl, icosanoyl, eicosanoyl, Henicosanoyl, Heneicosanoyl,docosanoyl, tricosanoyl, tetracosanoyl, pentacosanoyl, hexacosanoyl,heptacosanoyl, octacosanoyl, nonacosanoyl, triacontanoyl and the like.Examples of the unsaturated carboxylic acid acyl group include acryloyl,methacryloyl, crotonoyl, isocrotonoyl, butenoyl, butadienoyl, pentenoyl,hexenoyl, heptenoyl, octenoyl, nonenoyl, decenoyl, undecenoyl,dodecenoyl, tetradecenoyl, oleloyl, elaidinoyl, cyclopentanoyl,cyclohexanoyl, cycloheptanoyl, methylcyclopentanoyl,methylcyclohexanoyl, methylcycloheptanoyl, cyclopentenoyl,2,4-cyclopentadienoyl, cyclohexenoyl, 2,4-cyclohexadienoyl,cycloheptenoyl, methylcyclopentenoyl, methylcyclohexenoyl,methylcycloheptenoyl and the like.

The “alkyl group having 1 to 6 carbon atoms” means a linear or branchedalkyl group having 1 to 6 carbon atoms. Specific examples thereofinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl,2-pentyl, 3-pentyl, n-hexyl, 2-hexyl and the like.

The “alkoxyl group having 1 to 6 carbon atoms” means a linear orbranched alkoxyl group having 1 to 6 carbon atoms. Specific examplesthereof include a methoxy group, an ethoxy group, a propoxy group, abutoxy group, a pentyloxy group, a hexyloxy group, a methoxymethoxygroup, a methoxyethoxy group, a methoxypropoxy group, an ethoxyethoxygroup, an ethoxypropoxy group and the like.

The “hydroxyalkyl group having 1 to 6 carbon atoms” means a linear orbranched hydroxyalkyl group having 1 to 6 carbon atoms, wherein a partof the hydrogen atom in the alkyl group is substituted by a hydroxylgroup. Specific examples thereof include a hydroxymethyl group, a2-hydroxyethyl group, a 3-hydroxypropyl group, a 2-hydroxypropyl group,a 2-hydroxy-2-methylpropyl group, a 4-hydroxybutyl group, a3-hydroxybutyl group, a 2-hydroxybutyl group and the like.

The “haloalkyl group having 1 to 6 carbon atoms” means a linear orbranched haloalkyl group having 1 to 6 carbon atoms, wherein a part ofthe hydrogen atom in the alkyl group is substituted by a halogen atom,and the halogen atom is a fluorine atom, a chlorine atom, a bromine atomor an iodine atom. Specific examples thereof include a trifluoromethylgroup, a chloromethyl group, a bromomethyl group, a dichloromethylgroup, a difluoromethyl group, a trichloromethyl group, a 2-fluoroethylgroup, a 2-chloroethyl group, a 2-bromoethyl group, a 1,1-difluoroethylgroup, a 2,2,2-trifluoroethyl group, a 3-chloropropyl group, a3-iodopropyl group and the like.

The “haloalkoxyl group having 1 to 6 carbon atoms” means a linear orbranched haloalkoxyl group having 1 to 6 carbon atoms, wherein a part ofthe hydrogen atom in the alkoxy group is substituted by a halogen atom.Specific examples thereof include a trifluoromethoxy group, apentafluoroethoxy group, a 2-chloroethoxy group, a 2,2,2-trifluoroethoxygroup, a heptafluoro-n-propoxy group, a heptafluoro-i-propoxy group, a1,1,1,3,3,3-hexafluoro-2-propoxy group, a 3-fluoro-n-propoxy group, a1-chlorocyclopropoxy group, a 2-bromocyclopropoxy group, a3,3,4,4,4-pentafluoro-2-butoxy group, a noanfluoro-n-butoxy group, anonafluoro-2-butoxy group, a 5,5,5-trifluoro-n-pentyloxy group, a4,4,5,5,5-pentafluoro-2-pentyloxy group, a 3-chloro-n-pentyloxy group, a4-bromo-2-pentyloxy group, a 4-chlorobutyloxy group, a2-iodo-n-propyloxy group and the like.

The “halohydroxyalkyl group having 1 to 6 carbon atoms” means a linearor branched halohydroxyalkyl group having 1 to 6 carbon atoms, wherein apart of the hydrogen atom in the hydroxylalkyl group is substituted by ahalogen atom. Specific examples thereof include difluorohydroxymethyl,1,1-difluoro-2-hydroxyethyl, 2,2-difluoro-2-hydroxyethyl,1,1,2,2-tetrafluoro-2-hydroxyethyl group and the like.

The “forming a prodrug” in the “leaving group that can be bound to anamino group for forming a prodrug” means conversion of the targetcompound such that the compound which shows a small or no effect of thepresent invention shows the effect of the present invention upon removalof the leaving group in a medium and/or during culture. It meansformation of a temporary bond between the amino group and the leavinggroup in the compound showing the effect of the present invention, whichis removed in a medium and/or during culture.

The “leaving group that can be bound to an amino group for forming aprodrug” is not particularly limited as long as it is used in the fieldof synthetic organic chemistry. Examples thereof include a halogen atom(e.g., chlorine atom, bromine atom, iodine atom etc.), a sulfonyloxygroup (e.g., methanesulfonyloxy group, trifluoromethanesulfonyloxygroup, benzenesulfonyloxy group, p-toluenesulfonyloxy group etc.) andthe like.

When R³—CH is a Shiff base type amino-protecting group, R³ is a halogenatom, a hydroxy group, an aryl group, an acyl group having 2 to 30carbon atoms, an alkyl group having 1 to 6 carbon atoms, an alkoxylgroup having 1 to 6 carbon atoms, a hydroxyalkyl group having 1 to 6carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, ahaloalkoxyl group having 1-6 carbon atoms or a halohydroxyalkyl grouphaving 1 to 6 carbon atoms.

While the “hydroxyl-protecting group” is not particularly limited aslong as it is a hydroxyl-protecting group to be used in the field ofsynthetic organic chemistry, for example, an alkyl group having 1 to 6carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl,tert-butyl), a phenyl group, a trityl group, an aralkyl group having 7to 10 carbon atoms (e.g., benzyl, p-methoxybenzyl), a formyl group, analkyl-carbonyl group having 1 to 6 carbon atoms (e.g., acetyl,propionyl), a benzoyl group, an aralkyl-carbonyl group having 7 to 10carbon atoms (e.g., benzylcarbonyl), methoxymethyl, ethoxyethyl, a2-tetrahydropyranyl group, a 2-tetrahydrofuranyl group, substitutedsilyl group (e.g., trimethylsilyl, triethylsilyl, dimethylphenylsilyl,tert-butyldimethylsilyl, tert-butyldiethylsilyl), an alkenyl grouphaving 2 to 6 carbon atoms (e.g., 1-allyl) and the like can bementioned. Furthermore, a leaving group that can be bound to a hydroxygroup for forming a prodrug can also be mentioned.

The “forming a prodrug” in the “leaving group that can be bound to ahydroxy group for forming a prodrug” means conversion of the targetcompound such that the compound which shows a small or no effect of thepresent invention shows the effect of the present invention upon removalof the leaving group in a medium and/or during culture. It meansformation of a temporary bond between the hydroxy group and the leavinggroup in the compound showing the effect of the present invention, whichis removed in a medium and/or during culture.

The “leaving group that can be bound to a hydroxy group for forming aprodrug” is not particularly limited as long as it is used in the fieldof synthetic organic chemistry. Examples thereof include a halogen atom(e.g., chlorine atom, bromine atom, iodine atom etc.), a sulfonyloxygroup (e.g., methanesulfonyloxy group, trifluoromethanesulfonyloxygroup, benzenesulfonyloxy group, p-toluenesulfonyloxy group etc.) andthe like.

The ethanolamine analog is preferably one or plural selected from thegroup consisting of phosphoethanolamine (aka phosphoryl ethanolamine),monomethylethanolamine, dimethylethanolamine,N-acylphosphatidylethanolamine, phosphatidylethanolamine, andlysophosphatidylethanolamine.

The ethanolamine and/or ethanolamine analog to be used in the presentinvention may be in the form of a pharmaceutically acceptable salt.Examples of such salt when an acidic group such as a carboxyl group andthe like is present in the compound include salts with alkali metalssuch as ammonium salt, sodium, potassium and the like, salts withalkaline earth metals such as calcium, magnesium and the like, saltswith organic amines such as aluminum salt, zinc salt, triethylamine,morpholine, piperidine, dicyclohexylamine and the like, and salts withbasic amino acids such as arginine, lysine and the like. Examplesthereof when a basic group is present in the compound include salts withinorganic acids such as hydrochloric acid, sulfuric acid, phosphoricacid, nitric acid, hydrobromic acid and the like, salts with organiccarboxylic acids such as acetic acid, trifluoroacetic acid, citric acid,benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid,tannic acid, butyric acid, hibenzoic acid, pamoic acid, enanthic acid,decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid,mandelic acid, malic acid and the like, and salts with organic sulfonicacids such as methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and the like. Particularly preferred ishydrochloride.

The final concentration (concentration when in use) of ethanolamineand/or ethanolamine analogs and pharmaceutically acceptable saltsthereof in the medium can be determined to fall within any range as longas it can promote proliferation of pluripotent stem cells whilemaintaining an undifferentiated state under feeder-free conditions.While the final concentration can vary depending on the kind thereof, itis generally 1 μM to 1000 μM, preferably 5 μM to 200 μM, or 11 μM to 200μM. When it is less than 1 μM, the effect of promoting proliferation ofpluripotent stem cells while maintaining an undifferentiated state tendsto be weak. When it exceeds 1000 μM, proliferation of pluripotent stemcells may sometimes be suppressed. The concentration of at least oneselected from the group consisting of ethanolamine, ethanolamine analogsand pharmaceutically acceptable salts thereof in the medium is generally1 μM to 1000 μM, preferably 5 μM to 200 μM, or 11 μM to 200 μM. Whenplural kinds are used, the total amount thereof is set to fall withinthe above-mentioned range. However, it can be increased or decreased asappropriate depending on the number of the kinds thereof.

In the present invention, that at least one selected from the groupconsisting of ethanolamine and/or ethanolamine analogs andpharmaceutically acceptable salts thereof “promotes proliferation ofpluripotent stem cells while maintaining an undifferentiated state underfeeder-free conditions” means that the number of cells exceeding 100%can be obtained by adding at least one selected from the groupconsisting of ethanolamine and/or ethanolamine analogs andpharmaceutically acceptable salts thereof to the medium, wherein thenumber of pluripotent stem cells cultured under the same conditionsexcept that ethanolamine and/or ethanolamine analogs andpharmaceutically acceptable salts thereof are not added and in theabsence of a feeder cell is the standard (100%). Whether it promotesproliferation of pluripotent stem cells while maintaining anundifferentiated state under feeder-free conditions can be evaluated bya method using a known cell proliferation system, such as the methoddescribed in Examples and the like.

High concentration of β-mercaptoethanol is feared for its toxicity.Therefore, the concentration of β-mercaptoethanol used in the presentinvention is not more than 9 μM, more preferably not more than 7 μM,further preferably not more than 5 μM, as the final concentration whenin use. Furthermore, substantial or complete absence ofβ-mercaptoethanol is preferable. In the present invention, pluripotentstem cell can be stably proliferated while maintaining anundifferentiated state even when β-mercaptoethanol is not containedsubstantially.

The definition of the “substantial absence of β-mercaptoethanol” is asmentioned above.

The medium used in the present invention may be further added withalbumin. Addition of albumin affords a preservation stabilizing effecton the medium, as well as enhances the effect of ethanolamine to promoteproliferation of pluripotent stem cells while maintaining anundifferentiated state.

The “preservation stabilization” of the medium means alleviation of thetime-dependent deterioration of the medium during preservation of themedium (generally, about −80° C. to about 40° C.) and when in use. The“deterioration of the medium” in the present invention meansdeterioration of the function to proliferate pluripotent stem cellswhile maintaining an undifferentiated state, and the level thereof canbe evaluated by culturing pluripotent stem cells in said medium for agiven period, and counting the cell number, as described in thebelow-mentioned Examples. Use of a medium immediately after preparationbeing the standard, a smaller cell number after culture is evaluated asfurther deterioration of the medium.

The albumin to be used in the present invention is an animal-derivedserum albumin. Examples of the animal include, but are not particularlylimited to, rodents such as mouse, rat, hamster, guinea pig and thelike, experiment animals such as rabbit and the like, pets such as dog,cat and the like, domestic animals such as bovine, swine, goat, horse,sheep and the like, primates such as human, monkey, orangutan,chimpanzee and the like, and the like. When cells to be used forregenerative medicine are cultured, the albumin to be used in thepresent invention is preferably human albumin. Being “animal-derived”means that the amino acid sequence of the albumin is that of an animal.

The albumin used in the present invention may be isolated and purifiedfrom a biological sample of an animal (e.g., blood, plasma, serum etc.),or isolated and purified after production by gene recombinationtechnology. The preparation method of albumin is known. In addition,albumin may be a commercially available product and can be obtainedfrom, for example, Sigma-Aldrich Co. LLC. and the like.

The albumin used in the present invention is preferably obtained fromplasma of an animal (including human) or by gene recombinationtechnology.

The albumin used in the present invention carries fatty acid in anamount of preferably not more than 9 mg/g, more preferably not more than7 mg/g, further preferably not more than 2.2 mg/g.

When a medium added with albumin is used in the present invention, thefinal concentration (concentration when in use) of albumin in the mediumis not particularly limited as long as it affords a medium-stabilizingeffect, and enhances a promoting effect of at least one selected fromthe group consisting of ethanolamine, ethanolamine analogs andpharmaceutically acceptable salts thereof on the proliferation ofpluripotent stem cells while maintaining an undifferentiated state. Itis generally 0.1 g/l to 20 g/l, preferably 1 g/l to 8 g/l.

The medium to be used in the present invention may be further added withsulfated saccharides and/or a pharmaceutically acceptable salt thereof.Addition of sulfated saccharides in combination with ethanolamine to themedium affords a stabilizing effect on the medium, as well as enhancesthe effect of ethanolamine to promote proliferation of pluripotent stemcells while maintaining an undifferentiated state. When desired, pluralkinds of sulfated saccharides and/or pharmaceutically acceptable saltsthereof may be used.

In the present invention, the “sulfated saccharide” is sulfatedsubstance of saccharides. The “saccharide” is not particularly limitedas long as it is known in the technical field, or may be novel. Thesaccharide may be a natural product or synthesized product. The sulfatedsaccharides to be added to the medium of the present inventionpreferably include sulfated monosaccharide, sulfated disaccharide,sulfated polysaccharide, sulfated sugar alcohol and sulfated cyclitol.

The “monosaccharide” may be known in the technical field or novel. Thenumber of carbons constituting carbohydrate is not limited and may beany of, for example, tetrose, pentose, hexose, heptose and the like.Specific examples of the monosaccharide include glucose, galactose,mannose, talose, idose, altrose, allose, gulose, xylose, arabinose,rhamnose, fucose, fructose, ribose, deoxyribose, glucosamine,galactosamine, glucuronic acid, galacturonic acid and the like. Thesulfated monosaccharide is sulfated substance of these monosaccharides.

The “disaccharide” is a carbohydrate wherein two molecules of theaforementioned monosaccharide are bonded by a glycosidic bond to becomeone molecule, and may be known in the technical field or novel. Themanner of glycosidic bond is not particularly limited, and may be any ofα-1,2 bond, β-1,2 bond, α-1,3 bond, β-1,3 bond, α-1,4 bond, β-1,4 bond,α-1,5 bond, β-1,5 bond, α-1,6 bond, β-1,6 bond, α-1,α-1 bond, α-1,β-1bond, α-1,β-2 bond and the like. Specific examples of the disaccharideinclude sucrose, lactose, maltose, trehalose, cellobiose, maltitol andthe like. The sulfated disaccharide is sulfated substance of thesedisaccharides.

The polysaccharide is a carbohydrate wherein three or more molecules ofthe aforementioned monosaccharide are bonded by a glycosidic bond tobecome one molecule, and may be known in the technical field or novel.Polysaccharide may consist of only one kind of the aforementionedsaccharides, or two or more kinds thereof may be combined.Polysaccharide may be any of linear, branched and cyclic. Examples ofthe polysaccharide include amylose, amylopectin, glycogen, dextrin,α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, dextran, maltoheptaose,stachyose, maltotriose, pullulan, cellulose and a derivative thereof(e.g., hydroxyethylcellulose, hydroxypropylcellulose etc.), laminaran,curdlan, callose, mannan, glucomannan, galactomannan, xylan,glucuronoxylan, arabinoxylan, araban, galactan, galacturonan, chitin,chitosan, xyloglucan, pectic acid and pectin, alginic acid,arabinogalactan, glycosaminoglycan (e.g., dextran sulfate, heparansulfate, heparin, hyaluronic acid, chondroitin 4-sulfate, chondroitin6-sulfate, dermatan sulfate, ketaran sulfate etc.), guar gum, xanthangum, fucoidan, inulin and the like. The sulfated polysaccharide issulfated substance of these polysaccharides. Among the above-mentionedsaccharides, those already sulfated (e.g., dextran sulfate, heparansulfate, heparin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatansulfate, ketaran sulfate, fucoidan etc.) include the saccharidesthemselves. As the sulfated polysaccharide, dextran sulfate, sulfatedsubstance of cellulose (i.e., cellulose SO₃H), sulfated substance ofxanthan gum (i.e., xanthan gum SO₃H), fucoidan, sulfated substance ofalginic acid (i.e., alginate SO₃H), sulfated substance of inulin (i.e.,inulin SO₃H), sulfated substance of α-cyclodextrin (i.e., α-cyclodextrinSO₃H), sulfated substance of maltoheptaose (i.e., maltoheptaose SO₃H),sulfated substance of stachyose (i.e., stachyose SO₃H) and sulfatedsubstance of maltotriose (i.e., maltotriose SO₃H) are preferable, anddextran sulfate is particularly preferable.

The “sugar alcohol” is a compound produced by reducing the carbonylgroup of the aforementioned monosaccharide, and may be known in thetechnical field or novel. Examples of the sugar alcohol includeglycerol, erythritol, threitol, arabinitol, xylitol, sorbitol, mannitol,volemitol, perseitol and the like, and erythritol, xylitol and mannitolare preferable. The sulfated sugar alcohol is a sulfated substance ofthese sugar alcohols.

The “cyclitol” is polyhydroxycycloalkane, and also called cyclic sugaralcohol or cyclit. The cyclitol may be known in the technical field ornovel. While cyclitol is known to include many isomers, any isomer maybe used. While the number of carbons constituting the ring is notparticularly limited, a 6-membered ring is preferable. Examples of thecyclitol include inositol (1,2,3,4,5,6-cyclohexanehexaol), a derivativeof inositol (derivative wherein hydroxy group is substituted by aminogroup, ketone group, carboxyl group etc.) and the like. The sulfatedcyclitol is a sulfated substance of these cyclitols.

The sulfated saccharides to be added to the medium in the presentinvention may be in the form of a pharmaceutically acceptable salt.Examples of such salt include salts of a sulfate group etc. present inthe sulfated saccharides and a base. Specific examples thereof includealkali metal salts such as sodium salt, potassium salt and the like;alkaline earth metal salts such as calcium salt, magnesium salt and thelike; salts with inorganic base such as aluminum salt, ammonium salt andthe like; salts with organic base such as trimethylamine, triethylamine,pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine,triethanolamine, cyclohexylamine, dicyclohexylamine,N,N′-dibenzylethylenediamine and the like, which can be prepared from afree form by a conventional method.

As the pharmaceutically acceptable salt of the sulfated saccharides, asodium salt or potassium salt of a sulfate group is preferable. Examplesthereof include sucrose 8SO₃K, dextran sulfate Na (molecular weight5,000, 25,000, 500,000 etc.), cellulose SO₃Na, xanthan gum SO₃Na,alginic acid SO₃Na, inulin SO₃Na, α-cyclodextrin SO₃Na, erythritolSO₃Na, mannitol SO₃Na, myo-inositol 6SO₃K and the like, and dextransulfate Na is particularly preferable.

When a medium added with sulfated saccharides and/or a pharmaceuticallyacceptable salt thereof is used in the present invention, the finalconcentration (concentration when in use) of sulfated saccharides and/ora pharmaceutically acceptable salt thereof in the medium is notparticularly limited as long as the medium stabilizing effect isafforded and a promoting effect of at least one selected from the groupconsisting of ethanolamine, ethanolamine analogs and pharmaceuticallyacceptable salts thereof on the proliferation of pluripotent stem cellswhile maintaining an undifferentiated state is enhanced. It is generally1 to 1000 ng/ml, preferably 10 to 250 ng/ml. When plural kinds are used,the total amount thereof is set to fall within the above-mentionedrange. However, it can be increased or decreased as appropriatedepending on the kind.

The average molecular weight of sulfated saccharides or apharmaceutically acceptable salt thereof is not particularly limited,and varies depending on the kind of the sulfated saccharides to beemployed and the kind of the salt. It is generally 50 to 1,000,000,preferably 100 to 700,000, more preferably 300 to 500,000, mostpreferably 500 to 100,000. When the average molecular weight exceeds1,000,000, addition thereof at a concentration not less than a givenlevel tends to cause toxicity or suppression of cell proliferationseemingly due to the inhibition of cell adhesion and the like. Theaverage molecular weight can be measured by gel permeationchromatography and the like.

For example, the average molecular weight of the dextran sulfate Na isgenerally 1000 to 700,000, preferably 1000 to 300,000, more preferably1000 to 100,000, most preferably 2,500 to 7,500.

The medium to be used in the present invention may or may not containserum. When cells to be used for regenerative medicine are cultivated,in view of the possibility of serum being a virus infection source, andconcern about property difference between lots which causes inconsistentculture results and the like, culture is preferably performed underserum-free conditions.

The medium to be used in the present invention may or may not contain acomponent derived from a species different from the cell to be cultured.When human cells to be used for regenerative medicine are cultivated, acomponent derived from an animal other than human is preferably absentfrom the aspects of safety after transplantation.

As the basal medium to be used in the present invention, one known perse can be used depending on the kind of the pluripotent stem cells, andis not particularly limited as long as it does not inhibit proliferationof the pluripotent stem cells while maintaining an undifferentiatedstate. Examples thereof include DMEM, EMEM, IMDM (Iscove's ModifiedDulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's MediumF-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30,DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7,MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135,Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's BMOC-3Medium, E8 medium (Nature Methods, 2011, 8, 424-429), a mixed mediumthereof and the like. In addition, a medium altered for culture ofpluripotent stem cells, a mixture of the above-mentioned basal mediumand other medium, and the like may also be used.

The medium to be used in the present invention can further contain anadditive known per se. The additive is not particularly limited as longas it does not inhibit proliferation of pluripotent stem cells whilemaintaining an undifferentiated state. Examples thereof include growthfactor (e.g., insulin etc.), iron source (e.g., transferrin etc.),polyamines (e.g., putrescine etc.), mineral (e.g., sodium selenateetc.), saccharides (e.g., glucose etc.), organic acid (e.g., pyruvicacid, lactic acid etc.), amino acid (e.g., L-glutamine etc.), reducingagent (e.g., sodium thioglycolate), vitamins (e.g., ascorbic acid,d-biotin etc.), steroid (e.g., β-estradiol, progesterone etc.),antibiotic (e.g., streptomycin, penicillin, gentamicin etc.), bufferingagent (e.g., HEPES etc.) and the like. In addition, additives that havebeen conventionally used for culturing pluripotent stem cells can becontained as appropriate. The additive is preferably contained within aconcentration range known per se.

In the culture method of the present invention, a feeder cell may or maynot be used. When cells to be used for regenerative medicine arecultivated, from the aspect of safety after transplantation, a feedercell is preferably absent (feeder-free). Although not bound by anytheory, in the culture method of the present invention, proliferation ofpluripotent stem cells while maintaining an undifferentiated state canbe promoted even in the absence of a feeder cell, since ethanolaminegenerally secreted from a feeder cell is added to the medium.

When a feeder cell is not used, culture is preferably performed by usingan extracellular matrix or an active fragment thereof or an artificialproduct mimicking the functions thereof.

The extracellular matrix is not particularly limited as long as it isgenerally used for cell culture with the aim to improve adhesion betweenthe surface of a culture vessel and the cell. For example, known onessuch as laminin (laminin 511, laminin 332 etc.), fibronectin,vitronectin, collagen, elastin, adhesamine and the like can be used. Theactive fragment of an extracellular matrix only needs to be a fragmentthereof having a cell adhesion activity equivalent to that of theextracellular matrix, and known ones can be used. For example, E8fragment of laminin 511, E8 fragment of laminin 332 and the likedisclosed in JP-A-2011-78370 can be mentioned. The extracellular matrixand an active fragment thereof may be commercially available productsand available from, for example, (Life Technologies, BD Falcon,BioLamina) and the like. Two or more kinds of these extracellularmatrices and active fragments thereof may be used in combination. Also,a matrigel (trade name) which is a mixture of complicated basal laminacomponents containing protein and polysaccharides, that are extractedand purified from EHS sarcoma of mouse overproducing the basal lamina,may also be used. The extracellular matrix and an active fragmentthereof may be suspended in a suitable solution, and applied to acontainer suitable for cultivating cells.

An artificial product mimicking the function of extracellular matrix isnot particularly limited as long as it is generally used for culturingcells and, for example, known ones such as Synthemax (registered trademark) and Ultra-Web (registered trade mark) of Corning Incorporated,Hy-STEM series, polylysine and polyornithine of Sigma Aldrich Co., Ltd.and the like can be used.

The extracellular matrix or an active fragment thereof or an artificialproduct mimicking the functions thereof to be used in the presentinvention are preferably matrigel or laminin 511 or an active fragmentof laminin 511, more preferably an active fragment of laminin 511 (i.e.,E8 fragment of laminin 511).

In the culture method of the present invention, the cell seeding methodis not particularly limited, and may be colony seeding or singlecell-seeding. To produce pluripotent stem cells for regenerativemedicine at an industrial level, the work needs to be performed byplural workers under the conditions where procedures and schedule arerigorously managed. Therefore, single cell-seeding permitting rigorousadjustment of the seeding cell number is preferable.

For single cell-seeding, colonies of pluripotent stem cells aredissociated to single cells, and seeded in the medium. Single cellseeding can be performed by a method known per se. For example,cell-cell adhesion and cell-matrix adhesion are weakened with a celldetaching solution (trypsin solution etc.), and the cells are detachedfrom the matrix with a scraper (IWAKI, 9000-220 etc.) and the like (inthis state, the cells forming cell clusters are suspended in a solution,not complete single cells). The cells are thereafter dissociated bypipetting into single cells, and seeded in the medium. When seeding,ROCK inhibitor such as Y-27632 (Nacalai Tesque: 08945-84) and the likeis preferably added to the medium to ensure survival of the pluripotentstem cells. Since ROCK inhibitor is not necessary for the proliferationof pluripotent stem cells from the following day of the seeding, it ispreferably excluded from the medium.

Other culture conditions can be appropriately determined. For example,while the culture temperature is not particularly limited, it can beabout 30 to 40° C., preferably about 37° C. The CO₂ concentration can beabout 1 to 10%, preferably about 2 to 5%. The oxygen partial pressurecan be 1 to 10%.

In cell culture, medium change is sometimes necessary during the culturedue to the deterioration of medium components, accumulation of wasteproducts discharged from the cell and the like. In the culture method ofthe present invention, it is also possible to omit the medium change asshown in the Examples. For example, pluripotent stem cells can becultivated for 4 or more consecutive days (4 days, 5 days, 6 days etc.)without medium change.

In one embodiment, the present invention provides a culture method forthe proliferation of a pluripotent stem cell while maintaining anundifferentiated state, comprising a step of cultivating a pluripotentstem cell in a medium added with at least one selected from the groupconsisting of ethanolamine, ethanolamine analogs and pharmaceuticallyacceptable salts thereof, and added with sulfated saccharides and/or apharmaceutically acceptable salt thereof. The definitions ofethanolamine, ethanolamine analogs and pharmaceutically acceptable saltsthereof, and sulfated saccharides and a pharmaceutically acceptable saltthereof in the culture method are the same as those mentioned above, anduse concentrations thereof are also as mentioned above. In addition,other conditions relating to the culture method are also as mentionedabove.

Preservation Stabilizing Method for Medium for Proliferation ofPluripotent Stem Cells.

The present invention provides a preservation stabilizing method for amedium for the proliferation of a pluripotent stem cell, comprisingadding at least one selected from the group consisting of ethanolamine,ethanolamine analogs and pharmaceutically acceptable salts thereof, andadding sulfated saccharides and/or a pharmaceutically acceptable saltthereof (hereinafter to be also referred to as the stabilizing method ofthe present invention).

The “preservation stabilization” of the medium in the present inventionmeans alleviation of the time-dependent deterioration of the mediumduring preservation of the medium (generally, about −80° C. to 40° C.).The “deterioration of the medium” here means deterioration of thefunction to proliferate pluripotent stem cells while maintaining anundifferentiated state, and the level thereof can be evaluated byculturing pluripotent stem cells in said medium for a given period, andcounting the cell number, as described in the below-mentioned Examples.Use of a medium immediately after preparation being the standard, asmaller′cell number after culture is evaluated as further deteriorationof the medium.

The medium for the proliferation of pluripotent stem cells in thepresent invention is the same as the medium used in the above-mentionedculture method of the present invention, and preferably a medium for theproliferation of pluripotent stem cells while maintaining anundifferentiated state.

Ethanolamine and ethanolamine analogs and pharmaceutically acceptablesalts thereof to be added to the medium for preservation stability ofthe medium are the same as those used for the above-mentioned culturemethod of the present invention. The concentration of addition to themedium is the same as those in the above-mentioned culture method of thepresent invention. By adding ethanolamine and ethanolamine analogs andpharmaceutically acceptable salts thereof to the medium, the mediumafter addition can be stabilized.

The stabilizing method of the present invention further comprises a stepof adding sulfated saccharides and/or a pharmaceutically acceptable saltthereof. The sulfated saccharides and/or a pharmaceutically acceptablesalt thereof are the same as those used for the above-mentioned culturemethod of the present invention. The concentration of addition to themedium is the same as that in the above-mentioned culture method of thepresent invention. By adding at least one selected from the groupconsisting of ethanolamine, ethanolamine analogs and pharmaceuticallyacceptable salts thereof, and sulfated saccharides and/or apharmaceutically acceptable salt thereof in combination to the medium,the stability of the medium can be further enhanced.

According to the present invention, for example, when the medium ispreserved at room temperature (generally about 15 to 25° C.) for up toabout 8 days, after preparation of the medium, deterioration of themedium can be alleviated. Hence, the medium can be preserved for alonger term than before, and the present invention is useful for theproduction of pluripotent stem cells and the like.

Medium Additive for Proliferation of Pluripotent Stem Cells whileMaintaining an Undifferentiated State.

The component to be added to the basal medium in the above-mentionedculture method of the present invention can be a medium additive. Thatis, the present invention provides a medium additive for theproliferation of a pluripotent stem cell while maintaining anundifferentiated state, comprising at least one selected from the groupconsisting of ethanolamine, ethanolamine analogs and pharmaceuticallyacceptable salts thereof, and substantially free of β-mercaptoethanol orcontaining β-mercaptoethanol at a concentration of not more than 9 μMwhen in use (hereinafter to be also referred to as the medium additiveof the present invention).

Ethanolamine and ethanolamine analogs and pharmaceutically acceptablesalts thereof are the same as those used for the above-mentioned culturemethod of the present invention.

The amount of the medium additive of the present invention to be addedto a medium can be determined to fall within any range as long as it canpromote proliferation of pluripotent stem cells while maintaining anundifferentiated state when cultured under feeder-free conditions usingthe medium after preparation. It can be added such that the finalconcentration of ethanolamine and ethanolamine analogs andpharmaceutically acceptable salts thereof in the medium is generally 1μM to 1000 μM, preferably 5 μM to 200 μM, or 11 μM to 200 μM.

High concentration β-mercaptoethanol is feared for its toxicity.Therefore, the concentration of β-mercaptoethanol used in the presentinvention is not more than 9 μM, more preferably not more than 7 μM,further preferably not more than 5 μM, as the final concentration whenin use. Furthermore, substantial or complete absence ofβ-mercaptoethanol is preferable. In the present invention, pluripotentstem cell can be stably proliferated while maintaining anundifferentiated state even when β-mercaptoethanol is not containedsubstantially.

The definition of the “substantially free of β-mercaptoethanol” is asmentioned above.

The medium additive of the present invention may further containalbumin, sulfated saccharides and/or a pharmaceutically acceptable saltthereof. These are the same as those used for the above-mentionedculture method of the present invention.

Where necessary, the medium additive of the present invention maycontain other components other than the above-mentioned components.Examples of other components include, but are not particularly limitedto, additives generally used for the preparation of a medium, forexample, additives contained in the medium used in the above-mentionedproduction method of the present invention.

While the medium additive of the present invention may or may notcontain a component derived from a species different from the cell to becultured. When human cells to be used for regenerative medicine arecultivated, it is preferable that a component derived from an animalother than human not be contained.

Using the medium additive of the present invention, a medium capable ofstable proliferation for a long term while maintaining anundifferentiated state even in serum-free, feeder-free and singlecell-seeding culture can be prepared.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES

In the following Examples, proliferation effects of various testcompounds on human pluripotent stem cells were evaluated. As humanpluripotent stem cells, induced pluripotent stem cells (iPS cells, 201B7strain) purchased from iPS Academia Japan, Inc. were used, unlessparticularly specified. Cell culture was performed using a culturevessel (Becton, Dickinson and Company, Falcon culture petri dish orFalcon culture plate) coated with a basal lamina matrix under theconditions of 5% CO₂/37° C.

Various test compounds were added to a medium having the “E8”composition (disclosed in Nature Methods, 2011, 8, 424-429, which isincorporated herein by reference in its entirety) considered at presentto be the minimum composition for cultivating human pluripotent stemcells at given concentrations and used for culture, and the effectsthereof were studied. The medium was prepared by using Essential 8 (LifeTechnologies: A14666SA) considered to have the “E8” composition or amedium formulated to have an equivalent composition.

Reference Example 1 Medium Stabilizing Effect of Albumin

Human serum-derived albumin (Sigma-Aldrich Co. LLC: A1887) was added ata final concentration of 2.6 g/l, and the effect of albumin was examinedby studying the cell numbers after culture when used immediately afterpreparation for culture or when used after preservation at 4° C. for 3weeks after the preparation. The culture period was 1 week. 13,000viable cells per well were used for single cell seeding. As a basallamina matrix, a fragment containing an active domain of laminin 511,which was purchased from Osaka University, was applied at 5 μg/well.Y-27632 (final concentration 10 μM, Nacalai Tesque: 08945-84) was addedto the medium used for seeding. The cells were cultured in a medium freeof Y-27632 from the following day. Viable cell number was measured byTrypan Blue (Life Technologies: 15250-061) staining using ahemocytometer.

The average results of 3 series of experiments for each medium are shownin FIG. 1. When used immediately after preparation, equivalent cellproliferation was observed with or without albumin addition. When amedium after 3 weeks from preparation was used, cell proliferation wasscarcely found in an albumin-free medium, whereas obvious cellproliferation was observed in an albumin addition medium. The aboveresults reveal that E8 minimum composition medium shows remarkabledegradation of the property when kept under general use (preservation)conditions for 3 weeks, but addition of albumin can improve suchdeteriorative phenomenon. To conclude, albumin was found to contributeto the stabilization of a medium stored at 4° C.

Example 1 Proliferation Promoting Effect of Ethanolamine and CombinationEffect with Albumin

Ethanolamine (Sigma-Aldrich Co. LLC: E0135) was added at a finalconcentration of 6, 30, 150, 750 or 3,750 μM, and the medium was usedfor culture immediately after preparation, and the effect ofethanolamine was examined by studying the cell numbers after culture.The culture period was 1 week. To study the effect of combination withalbumin, human serum-derived albumin (Sigma-Aldrich Co. LLC: A1887) wasfurther added at a final concentration of 2.6 g/l to the above-mentionedethanolamine addition medium, and a similar examination was performed.13,000 viable cells per 1 well were used for single cell-seeding. As abasal lamina matrix, a fragment containing the active domain of laminin511, which was purchased from Osaka University, was applied at 5μg/well. Y-27632 was added (final concentration 10 μM, Nacalai Tesque:08945-84) to the medium used for seeding. The cells were cultured in amedium free of Y-27632 from the following day. The number of viablecells was measured by Trypan Blue (Life Technologies: 15250-061)staining using a hemocytometer.

The experiment was performed in 3 series for each medium, and theaverage results are shown in FIG. 2. The values are shown as relativevalues to the ethanolamine non-addition group (0 μM). Ethanolamine wasfound to show a proliferation-promoting effect in a relatively wideconcentration range. It was found that a proliferation suppressiveeffect conversely appears in a high concentration range. It was foundthat combination with albumin further enhances the proliferationpromoting effect of ethanolamine, resists a proliferation suppressiveeffect in a high concentration range, and shows a proliferationpromoting effect.

Example 2 Effect of Dextran Sulfate

Human serum derived albumin (Sigma-Aldrich Co. LLC: A1887) was added toa medium (final concentration 2.6 g/l), ethanolamine (finalconcentration 30 μM) was added singly thereto, dextran sulfate sodium(Wako Pure Chemical Industries, Ltd., final concentration 50 ng/ml) wasfurther added to the medium, and a further proliferation promotingeffect of a combination of ethanolamine and dextran sulfate sodium wasverified. Culture was performed for about 3 weeks, which included twopassages, and the cumulative viable cell increase rate for the totalculture period was calculated. 13,000 viable cells per 1 well were usedfor single cell-seeding. As a basal lamina matrix, a fragment containingthe active domain of laminin 511, which was purchased from OsakaUniversity, was applied at 5 μg/well. Y-27632 was added (finalconcentration 10 μM, Nacalai Tesque: 08945-84) to the medium used forseeding. The cells were cultured in a medium free of Y-27632 from thefollowing day. When passaged, cells were detached by TrypLETm Select(Life Technologies: 12563-011), 13,000 viable cells were seeded again inY-27632 added medium, and cultured in a medium free of Y-27632 from thefollowing day. The number of viable cells was measured by Trypan Blue(Life Technologies: 15250-061) staining using a hemocytometer.

The experiment was performed in 3 series for each medium, and theaverage results are shown in FIG. 3. It was found that the cell increaserate was higher when dextran sulfate sodium was combined than the singleaddition of ethanolamine.

Both ethanolamine and dextran sulfate sodium, which showed the highestincrease rate, were added to a medium, and culture was performed forabout one month. Alkaline phosphatase staining was performed to confirmmaintenance of undifferentiated state. The results of staining with analkaline phosphatase staining kit (Sigma-Aldrich Co. LLC: 86-R) areshown in FIG. 4. iPS cell colonies in the whole well were stained, whichconfirmed that long-term culture in an E8 minimum composition mediumadded with albumin, ethanolamine, dextran sulfate sodium resulted in theproliferation of iPS cells while maintaining an undifferentiated state.

Example 3 Proliferation Promoting Effect of Ethanolamine—Results ofCulture Using Matrigel

Ethanolamine (Sigma-Aldrich Co. LLC: E0135) was added at a finalconcentration of 6, 30, 150, 750 or 3,750 μM, and the medium was usedfor culture immediately after preparation, and the effect ofethanolamine was examined by studying the cell numbers after culture.The culture period was 8 days. 100,000 cells per 1 well were used forsingle cell-seeding. As a basal lamina matrix, matrigel (Japan BectonDickinson) was applied. Y-27632 was added (final concentration 10 TIM,Nacalai Tesque: 08945-84) to the medium used for seeding. The cells werecultured in a medium free of Y-27632 from the following day.

The experiment was performed in 3 series for each medium, and theaverage results are shown in FIG. 5. In the same manner as in Example 1,the results showing that ethanolamine affords a proliferation promotingeffect in a wide concentration range were obtained. Therefore, it wasfound that the proliferation promoting effect of ethanolamine is notlimited to culture using laminin 511.

Example 4 Effect of Combination of Ethanolamine and Dextran SulfateSodium—Results of Culture Using Matrigel (1) Effect in Medium ContainingAlbumin.

Essential 8 medium added with human serum derived albumin (finalconcentration 2.6 g/l) and ethanolamine (final concentration 30 μM)(control), and the medium added with dextran sulfate sodium at a givenconcentration were prepared, and the effect of combination ofethanolamine and dextran sulfate sodium was verified. The culture periodwas 6 to 12 days. For single cell-seeding, 40,000 cells per 1 well wereseeded on a 12-well culture plate coated with matrigel. Y-27632 wasadded (final concentration 10 μM, Nacalai Tesque: 08945-84) to themedium used for seeding. The cells were cultured in a medium free ofY-27632 from the following day. For seeding as colony, the cells diluted2.5- to 3.5-fold of the original culture per well were plated on a 6well culture plate coated with matrigel. In this case, Y-27632 was notadded to the medium to be used for seeding. The medium change wasperformed every 2 to 3 days.

The evaluation criteria of cell proliferation were as follows.

⊙: cell number is not less than 120% of that of control◯: cell number is not less than 100% and less than 120% of that ofcontrol−: cell number is not less than 50% and less than 100% of that ofcontrolx: cell number is not more than 50% of that of control

The experiment was performed in 3 series for each medium, and theresults are shown in Table 1. Higher cell proliferation was observedwhen dextran sulfate sodium was added. Therefore, it was found that thecell proliferation promoting effect afforded by a combination ofethanolamine and dextran sulfate sodium is not limited to culture usinglaminin 511. It was also found that the effect was exhibited even whenseeded as colony, and the effect is not limited to single cell-seeding.

TABLE 1 final concentration (ng/ml) of dextran sulfate sodium 1 10 50100 single ◯ ◯ ⊙ not cell- evaluated seeding colony not evaluated ⊙ not⊙ seeding evaluated

(2) Effect of Omission of Medium Change.

In the evaluation of the above-mentioned (1), culture was performed in amedium added with dextran sulfate sodium (final concentration 10 ng/ml)without medium change, and whether medium change can be omitted byadding ethanolamine and dextran sulfate sodium in combination to themedium was verified. The culture period was 6 days.

The experiment was performed in 3 series for each medium, and theresults are shown in FIG. 6. When medium change was not performed, highcell proliferation was found by the addition of dextran sulfate sodium.The effect thereof was close to that afforded by medium change with amedium free of dextran sulfate sodium. Therefore, it was found that acombination of ethanolamine and dextran sulfate sodium provides aneffect of omitting medium change.

(3) Stabilizing Effect in Medium without Albumin.

Essential 8 medium added with ethanolamine (final concentration 30 μM)alone, the medium added with ethanolamine (final concentration 30 μM)and dextran sulfate sodium (Wako Pure Chemical Industries, Ltd., finalconcentration 50 ng/ml), and the medium added with human serum derivedalbumin (final concentration 2.6 g/l) alone were prepared, and a mediumstabilizing effect of a combination of ethanolamine and dextran sulfatesodium was verified. Each medium was left standing at room temperaturefor 8 days after preparation and used for culture. The culture periodwas 8 days. 100,000 cells per 1 well were used for single cell-seedingon a 6-well culture plate coated with matrigel. Y-27632 was added (finalconcentration 10 μM, Nacalai Tesque: 08945-84) to the medium used forseeding. The cells were cultured in a medium free of Y-27632 from thefollowing day.

The experiment was performed in 3 series for each medium, and theaverage results are shown in FIG. 7. The effect afforded by adding bothethanolamine and dextran sulfate sodium was equal to that afforded byadding albumin. Therefore, it was found that a combination ofethanolamine and dextran sulfate sodium provides a medium stabilizingeffect at room temperature, and has a possibility of replacing orreducing albumin widely used for medium.

Example 5 Effect of Ethanolamine Analogs (1) Effect of O-PhosphorylEthanolamine (Aka Phosphoethanolamine).

To a medium added with human serum-derived albumin (Sigma-Aldrich Co.LLC.: A1887) at a final concentration of 2.6 g/l was added O-phosphorylethanolamine (Sigma-Aldrich Co. LLC.: P0503-25G) at a finalconcentration of 6, 30, 150 or 750 μM, and used for the culture from thenext day of preparation. After the culture, the cell number was countedto examine the effect of O-phosphoryl ethanolamine. The culture periodwas set to 1 week. 13,000 viable cells per well were used for singlecell-seeding. As a basal lamina matrix, a fragment containing the activedomain of laminin 511, which was purchased from Osaka University, wasapplied at 4.8 μg/well. Y-27632 was added (final concentration 10 μM,Nacalai Tesque: 08945-84) to the medium used for seeding. The cells werecultured in a medium free of Y-27632 from the following day. The numberof viable cells was measured by Cell Viability autoanalyzer ViCELL™ XR(BECKMAN COULTER).

The experiment was performed in 3 series for each medium, and theaverage results are shown in FIG. 8. The values are shown as relativevalues to the O-phosphoryl ethanolamine non-addition group (0 μM).O-phosphoryl ethanolamine was found to show a proliferation-promotingeffect in a relatively wide concentration range.

(2) Effect of 2-(Methylamino)Ethanol.

To a medium added with human serum-derived albumin (Sigma-Aldrich Co.LLC.: A1887) at a final concentration of 2.6 g/l was added2-(methylamino)ethanol (Sigma-Aldrich Co. LLC.: 471445-25ML) at a finalconcentration of 6, 30, 150 or 750 μM, and used for the culture from thenext day of preparation. After the culture, the cell number was countedto examine the effect of 2-(methylamino)ethanol. The culture period wasset to 1 week. 13,000 viable cells per well were used for singlecell-seeding. As a basal lamina matrix, a fragment containing the activedomain of laminin 511, which was purchased from Osaka University, wasapplied at 4.8 μg/well. Y-27632 was added (final concentration 10 μM,Nacalai Tesque: 08945-84) to the medium used for seeding. The cells werecultured in a medium free of Y-27632 from the following day. The numberof viable cells was measured by Cell Viability autoanalyzer ViCELL™ XR(BECKMAN COULTER).

The experiment was performed in 3 series for each medium, and theaverage results are shown in FIG. 9. The values are shown as relativevalues to the 2-(methylamino)ethanol non-addition group (0 μM).2-(Methylamino)ethanol was found to show a proliferation-promotingeffect in a relatively wide concentration range.

(3) Effect of 2-Dimethylaminoethanol.

To a medium added with human serum-derived albumin (Sigma-Aldrich Co.LLC.: A1887) at a final concentration of 2.6 g/l was added2-dimethylaminoethanol (Sigma-Aldrich Co. LLC.: 471453-100 ml) at afinal concentration of 6, 30, 150 or 750 μM, and used for the culturefrom the next day of preparation. After the culture, the cell number wascounted to examine the effect of 2-dimethylaminoethanol. The cultureperiod was set to 1 week. 13,000 viable cells per well were used forsingle cell-seeding. As a basal lamina matrix, a fragment containing theactive domain of laminin 511, which was purchased from Osaka University,was applied at 4.8 μg/well. Y-27632 was added (final concentration 10μM, Nacalai Tesque: 08945-84) to the medium used for seeding. The cellswere cultured in a medium free of Y-27632 from the following day. Thenumber of viable cells was measured by Cell Viability autoanalyzerViCELL™ XR (BECKMAN COULTER).

The experiment was performed in 3 series for each medium, and theaverage results are shown in FIG. 10. The values are shown as relativevalues to the 2-dimethylaminoethanol non-addition group (0 μM). It wasfound that 2-dimethylaminoethanol shows a proliferation promoting effectin a relatively wide concentration range, but a proliferationsuppressive effect appears in a high concentration range.

(4) Effect of Ethanolamine Hydrochloride.

To a medium added with human serum-derived albumin (Sigma-Aldrich Co.LLC.: A1887) at a final concentration of 2.6 g/l was added ethanolaminehydrochloride (Tokyo Chemical Industry Co., Ltd.: A0298) at a finalconcentration of 6, 30, 150 or 750 μM, and used for the culture from thenext day of preparation. After the culture, the cell number was countedto examine the effect of ethanolamine hydrochloride. The culture periodwas set to 1 week. 13,000 viable cells per well were used for singlecell-seeding. As a basal lamina matrix, a fragment containing the activedomain of laminin 511, which was purchased from Osaka University, wasapplied at 4.8 μg/well. Y-27632 was added (final concentration 10 μM,Nacalai Tesque: 08945-84) to the medium used for seeding. The cells werecultured in a medium free of Y-27632 from the following day. The numberof viable cells was measured by Cell Viability autoanalyzer ViCELL™ XR(BECKMAN COULTER).

The experiment was performed in 3 series for each medium, and theaverage results are shown in FIG. 11. The values are shown as relativevalues to the ethanolamine hydrochloride non-addition group (0 μM).Ethanolamine hydrochloride was found to show a proliferation-promotingeffect in a relatively wide concentration range.

Example 6 Influence of Oleic Acid

To a solution (25%, 40 ml) of human serum albumin (Nova Biologics) insaline was added phosphate buffer (pH 7.2, 40 ml). Furthermore, asuspension of activated carbon (5 g, manufactured by Wako Pure ChemicalIndustries, Ltd.) heated at 200° C. for 30 min in advance in phosphatebuffer (20 ml) was added to 100 ml. After stirring at 4° C. for 3 hr,the mixture was centrifuged at 4° C., 11,900 rpm for 20 min. Sedimentedactivated carbon was removed by decantation, and the reaction mixturewas filtered with a 0.22 μm syringe filter, whereby human serum albuminwith fatty acid removal treatment was obtained. Then, oleic acid wasadded to the obtained human serum albumin with fatty acid removaltreatment to prepare human serum-derived albumin having a fatty acidcarried amount of 2.2, 6.5, 21.7 mg/g, and each albumin was added to themedium to a final concentration of 2.6 g/l (final concentration of oleicacid in the medium 20, 60, 200 μM, respectively). Ethanolamine (finalconcentration 30 μM) was added to each medium. Using each medium, thecells were cultured for one week, and the cell number after culture wasconfirmed to study the influence of oleic acid. 13,000 viable cells perwell were used for single cell-seeding. As a basal lamina matrix, afragment containing the active domain of laminin 511, which waspurchased from Osaka University, was applied at 4.8 μg/well. Y-27632 wasadded (final concentration 10 μM, Nacalai Tesque: 08945-84) to themedium used for seeding. The cells were cultured in a medium free ofY-27632 from the following day. The number of viable cells was measuredby Cell Viability autoanalyzer ViCELL™ XR (BECKMAN COULTER).

The results of culture using each medium are shown in FIG. 12. It wasfound that cell proliferation was suppressed along with an increasingamount of oleic acid carried by albumin.

INDUSTRIAL APPLICABILITY

According to the present invention, pluripotent stem cells can be stablyand efficiently proliferated, and can be stably proliferated for a longterm while maintaining an undifferentiated state even in serum-free,feeder-free and single cell-seeding culture, and therefore, it is usefulin the field of regenerative medicine and the like.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

As used herein the words “a” and “an” and the like carry the meaning of“one or more.”

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

All patents and other references mentioned above are incorporated infull herein by this reference, the same as if set forth at length.

1. A culture medium for a proliferation of a pluripotent stem cell whilemaintaining an undifferentiated state, wherein said medium comprises analbumin, at least one member selected from the group consisting ofethanolamine, an ethanolamine analog, and a pharmaceutically acceptablesalt thereof, and wherein said medium is substantially free ofβ-mercaptoethanol or contains β-mercaptoethanol in a concentration ofnot more than 9 μM, and wherein the concentration of said at least onemember selected from the group consisting of ethanolamine, anethanolamine analog, and a pharmaceutically acceptable salt thereof insaid medium is 11 μM to 200 μM, based on the volume of said medium. 2.The culture medium according to claim 1, which is substantially free ofβ-mercaptoethanol.
 3. The culture medium according to claim 1, whereinthe amount of fatty acid carried by said albumin in said medium is notmore than 9 mg/g, based on the weight of said albumin.
 4. The culturemedium according to claim 1, wherein the amount of fatty acid carried bysaid albumin in said medium is not more than 2.2 mg/g, based on theweight of said albumin.
 5. The culture medium according to claim 1,which further comprises a sulfated saccharide and/or a pharmaceuticallyacceptable salt thereof.
 6. A culture method for proliferation of apluripotent stem cell while maintaining an undifferentiated state,comprising cultivating said pluripotent stem cell in a medium whichcomprises an albumin, at least one member selected from the groupconsisting of ethanolamine, an ethanolamine analog, and apharmaceutically acceptable salt thereof, and wherein said medium issubstantially free of β-mercaptoethanol or contains β-mercaptoethanol ina concentration of not more than 9 μM, and wherein the concentration ofsaid at least one member selected from the group consisting ofethanolamine, an ethanolamine analog and a pharmaceutically acceptablesalts thereof in said medium is 11 μM to 200 μM, based on the volume ofsaid medium.
 7. The method according to claim 6, wherein said medium issubstantially free of β-mercaptoethanol.
 8. The method according toclaim 6, wherein the amount of fatty acid carried by said albumin insaid medium is not more than 9 mg/g, based on the weight of saidalbumin.
 9. The method according to claim 6, wherein the amount of fattyacid carried by said albumin in said medium is not more than 2.2 mg/g,based on the weight of said albumin.
 10. The method according to claim6, wherein said medium further comprises sulfated saccharide and/or apharmaceutically acceptable salt thereof.
 11. The method according toclaim 6, wherein said cultivating is performed in the absence of afeeder cell.
 12. The method according to claim 11, wherein saidcultivating is performed by using an extracellular matrix or an activefragment thereof, or an artificial product mimicking the functionthereof.
 13. The method according to claim 6, wherein said cultivatingis performed by single cell-seeding.
 14. The method according to claim6, wherein said pluripotent stem cell is an embryonic stem cell (EScell) or an induced pluripotent stem cell (iPS cell).
 15. A mediumadditive for proliferation of a pluripotent stem cell while maintainingan undifferentiated state, comprising an albumin, at least one memberselected from the group consisting of ethanolamine, an ethanolamineanalog, and a pharmaceutically acceptable salts thereof, and which issubstantially free of β-mercaptoethanol or contains β-mercaptoethanol ina concentration of not more than 9 μM when in use, wherein theconcentration of said at least one member selected from the groupconsisting of ethanolamine, an ethanolamine analog, and apharmaceutically acceptable salt thereof in said medium is 11 μM to 200μM, based on the volume of said medium.
 16. The medium additiveaccording to claim 15, which is substantially free of β-mercaptoethanol.17. The medium additive according to claim 15, wherein the amount offatty acid carried by said albumin is not more than 9 mg/g based on theweight of said albumin.
 18. The medium additive according to claim 15,wherein the amount of fatty acid carried by said albumin is not morethan 2.2 mg/g, based on the weight of said albumin.
 19. The mediumadditive according to claim 15, further comprising sulfated saccharideand/or a pharmaceutically acceptable salt thereof.
 20. A preservationstabilizing method for a medium for a proliferation of a pluripotentstem cell, comprising adding to a medium, an albumin, at least onemember selected from the group consisting of ethanolamine, anethanolamine analog, and a pharmaceutically acceptable salt thereof, anda sulfated saccharide and/or a pharmaceutically acceptable salt thereof,wherein the concentration of said at least one member selected from thegroup consisting of ethanolamine, an ethanolamine analog, and apharmaceutically acceptable salt thereof in said medium is 11 μM to 200μM, based on the volume of said medium.
 21. The method according toclaim 20, wherein said medium is substantially free ofβ-mercaptoethanol.
 22. The method according to claim 20, wherein theamount of fatty acid carried by said albumin is not more than 9 mg/g,based on the weight of said albumin.
 23. The method according to claim20, wherein the amount of fatty acid carried by said albumin is not morethan 2.2 mg/g, based on the weight of said albumin.